Methods for regulating neural differentiation

ABSTRACT

Methods of producing populations of predominantly astrocytes, neurons or oligodendrocytes are provided. In addition, methods of treating mammals having astroglial tumors, oligodendrocyte tumors, or neuronal tumors are provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional application of, and claims the benefitof priority under 35 U.S.C. §121 to, U.S. application Ser. No.13/213,848, filed on Aug. 19, 2011, which claims the benefit of priorityunder 35 U.S.C. §119(e) to U.S. application Ser. No. 61/471,368, filedApr. 4, 2011, and U.S. application Ser. No. 61/375,154, filed Aug. 19,2010.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under NS048187 awardedby the National Institutes of Health. The Government has certain rightsin the invention.

TECHNICAL FIELD

This document provides methods related to differentiation of stem cells(e.g., neural stem cells). For example, this document provides methodsfor differentiating stem cells into predominantly astrocytes orpredominantly neurons and oligodendrocytes.

BACKGROUND

Regulation of gene expression during development occurs at multiplelevels as indicated by the recent identification of numerouscis-regulatory sequences, transcription complexes, microRNAs (miRNAs),and additional classes of non-coding RNAs. miRNAs are small,single-stranded RNAs that play important roles in proliferation,differentiation, tumorigenesis, and apoptosis via their ability tosilence target genes. miRNAs function primarily as repressors of geneexpression, typically acting at the level of translational regulation ofgene expression. miRNAs can also promote mRNA degradation and, inquiescent cells, activate translation. The ability of miRNAs to regulatelarge sets of target genes may provide “developmental switches” duringlineage differentiation of various cell types including stem cells.

SUMMARY

In one aspect, a method of producing a population of predominantlyastrocytes is provided. Such a method typically includes overexpressingmiRNA-410 in a population of stem cells or downstream precursor cells.Generally, the overexpressing miRNA-410 results in differentiation ofthe cells into a population of predominantly astrocytes.

In certain embodiment, overexpressing miRNA-410 comprises introducing anucleic acid molecules comprising a promoter operably linked to a DNAsequence encoding miRNA-410. In some embodiments, the promoter is aninducible promoter. In some embodiments, the method is performed invitro. In some embodiments, the stem cells are neural stem cells.

In another aspect, a method of producing a population of predominantlyastrocytes is produced. Such methods typically include contacting apopulation of stem cells or downstream precursor cells with miRNA-410.Generally, the contacting step results in differentiation of the cellsinto a population of predominantly astrocytes.

In certain embodiments, the contacting further comprises using alipid-based delivery system. In certain embodiments, the method isperformed in vitro. In some embodiments, the stem cells are neural stemcells.

In yet another aspect, a method of producing a population ofpredominantly neurons and oligodendrocytes is provided. Such a methodtypically includes inhibiting the expression or activity of miRNA-410 ina population of stem cells or downstream precursor cells. Generally, theinhibiting expression or activity of miRNA-410 results indifferentiation of the cells into a population of predominantly neuronsand oligodendrocytes.

In certain embodiments, the inhibiting comprises using (e.g., contactingcells with) an antagomir specific for miRNA-410. In certain embodiments,the inhibiting comprises using (e.g., contacting cells with) a2-O-methyl oligoribonucleotide (2-O-Me-RNA) specific for miRNA-410. Incertain embodiments, inhibiting comprises using (e.g., contacting cellswith) a morpholino oligonucleotide complementary to miRNA-410. Incertain embodiments, the inhibiting comprises using (e.g., contactingcells with) an miRNA sponge. In certain embodiments, the inhibitingcomprises using (e.g., contacting cells with) LNA oligonucleotides.

In some embodiments, the method is performed in vitro. In someembodiments, the stem cells are neural stem cells. In some embodiments,the method further includes contacting the cells with noggin.

In still another aspect, a method for identifying a compound thatinhibits or induces the activity of miRNA-410 is provided. Such a methodtypically includes contacting a nucleic acid molecule with a testcompound in the presence of miRNA-410, wherein the nucleic acid moleculecomprises a promoter operably linked to nucleic acid encoding amiRNA-410 binding site operably linked to nucleic acid encoding areporter protein. Generally, an increase in the amount of reporterprotein is indicative of a compound that inhibits the activity ofmiRNA-410, and a decrease in the amount of reporter protein isindicative of a compound that increases the activity of miRNA-410. Incertain embodiments, the nucleic acid molecule is comprised within ahost cell.

In yet another aspect, a method of treating an animal having anastroglial tumor is provided. Such a method typically includescontacting the tumor with a compound that inhibits the expression oractivity of miRNA-410, wherein the contacting promotes differentiationof cells in the astroglial tumor. In certain embodiments, the contactingcomprises direct injection of the compound into the tumor.Representative compounds that inhibit the expression or activity ofmiRNA-410 include, for example, an antagomir specific for miRNA-410, amorpholino oligonucleotide complementary to miRNA-410, 2-O-methyloligoribonucleotide (2-O-Me-RNA) specific for miRNA-410, LNAoligonucleotides, and the sponge. Representative tumors include aglioma, astrocytoma (protoplasmic, gemistocytic, fibrillary), pilocyticastrocytoma, subependymal astrocytoma, pleomorphic xanthoastrocytoma,and neurofibromas.

In still another aspect, a method of treating an animal having aneuronal tumor is provided. Such a method typically contacting the tumorwith miRNA-410 or a compound that increases the expression or activityof miRNA-410, wherein the contacting promotes differentiation of cellsin the neuronal tumor. In certain embodiments, the contacting comprisesdirect injection of the miRNA-410 or the compound into the tumor. Arepresentative compound that increases the expression or activity ofmiRNA-410 is a nucleic acid molecule that includes a promoter operablylinked to a miRNA-410 DNA. Representative neuronal tumors include, forexample, neuroblastoma, medulloblastoma, retinoblastoma, gangliocytoma,neurocytoma, and ependymoblastoma.

In still another aspect, a method of treating an animal having anoligodendrocyte tumor is provided. Such a method typically contactingthe tumor with miRNA-410 or a compound that increases the expression oractivity of miRNA-410, wherein the contacting promotes differentiationof cells in the oligodendrocyte tumor. In certain embodiments, thecontacting comprises direct injection of the miRNA-410 or the compoundinto the tumor. A representative compound that increases the expressionor activity of miRNA-410 is a nucleic acid molecule that includes apromoter operably linked to a miRNA-410 DNA. Representativeoligodendrocyte tumors include, for example, oligoastrocytomas andoligodendrogliomas.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used to practicethe invention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the hairpin structure of miR-410.

FIG. 2 is a bar graph demonstrating that miR-410 expression isdown-regulated 16-fold after noggin expression in the adultsubventricular zone (SVZ).

FIG. 3 is a series of images demonstrating expression of miR-410 (3A) inthe CNS in E11.5 mouse embryos. In the adult mouse brain, it isexpressed at high levels in the SVZ neural stem cell zone (3C).

FIG. 4 is a schematic depicting a miR-410 over-expression vector (4A)and an expression construct where the ubiquitin promoter drivesexpression of a “sponge” in which six miR-410 binding sites areexpressed downstream of mCherry (4B).

FIG. 5 is a series of images depicting mouse embryonic stem (ES) celllines stably over-expressing miR-410 (indicated by mCherry) express theESC marker Oct4 in control ES cell culture conditions.

FIG. 6 is a series of images demonstrating widespread differentiation ofTuj1 positive neurons. (6A-B) After 6 days in vitro, control D3 cellsdifferentiated widely into neurons (Tuj1+) whereas neuronaldifferentiation was abrogated in over-expressing ES cell line 20 (6C-D)and line 24 (6E-F). Tuj1: FITC; nuclei: Hoechst 33258.

FIG. 7 is a series of images depicting TUNEL staining of control (7A)and miRNA-410 overexpressing lines 20 (7B) and 24 (7C).

FIG. 8 is a set of bar graphs demonstrating that (8A) miRNA-410 isover-expressed 20-fold in line #20 and it is expressed 7-fold higherthan in control cells in line #24. (8B) q-RT-PCR analysis showed thatexpression of an ESC marker (Oct4), an endoderm lineage marker (FoxA2),and a mesoderm lineage marker (Brachyury) were not affected, whileneural lineage markers (Sox3 and Tuj1) were down-regulated byover-expression of miRNA-410.

FIG. 9 is a photograph of spheres produced during differentiation ofneuronal stem cells illustrating the persistent expression of mCherryand, therefore, the transgene.

FIG. 10 illustrates the presence of astrocytes and neurons followingover-expression of miRNA-410 (Panels C and D, respectively) andfollowing the use of the microRNA sponge to inhibit miRNA-410 (Panels Eand F, respectively) compared to control cells (Panels A and B,respectively).

FIG. 11 is a set of bar graphs showing that the number of Tuj1-positiveneurons was increased by miRNA-410 knock-down and decreased by theover-expression of miRNA-410. The number of GFAP-positive astrocytes wassignificantly decreased in cells transfected with miRNA-410 sponge,compared with controls. Furthermore, miRNA-410 over-expression rescuedthe increase in Tuj1-positive neurons and the reduction in GFAP-positivecells produced by noggin.

FIG. 12 is a series of images demonstrating that over-expression ofmiRNA-410 strikingly reduces the number of Sox3-positive neuralprecursors compared to the parental D3 ES cells.

FIG. 13 is a series of images demonstrating that over-expression ofmiRNA-410 strikingly reduces the number of Tuj1-positive neuronscompared to the parental D3 ES cells.

FIG. 14 illustrates the morphology of oligodendrocytes derived from theSVZ in control culture conditions (A), following miRNA-410over-expression (B), and exposure to the sponge (C,D). The number andmaturity of MBP+ oligodendrocytes was increased in the presence of thesponge (C), and particularly in the presence of noggin and the sponge.

FIG. 15 is a graph showing the relative amount of miR-410 inglioblastomas.

FIG. 16 is a graph showing reporter gene expression using wild type(dark gray bars) or mutant (light gray bars) miR-410 recognitionsequences from the Elavl4/HuD, Tcf4, Fgf7, Smad7, Sox1, Zfx, and Msl2genes.

DETAILED DESCRIPTION

This disclosure describes a novel regulatory role for miRNA-410. Asdescribed herein, miRNA-410 plays a critical role in controlling thedifferentiation of stem cells (e.g., neural stem cells) or downstreamprecursor cells (e.g., neuronal or glial precursor cells) intopredominantly astrocytes or predominantly neurons and oligodendrocytes.

MicroRNAs

MicroRNAs (miRNAs) are short (20-24 nt) non-coding RNAs that play a rolein post-transcriptional regulation of gene expression in multicellularorganisms by affecting both the stability and translation of mRNAs.miRNAs can be transcribed by RNA polymerase II as part of capped andpolyadenylated primary transcripts (pri-miRNAs) that can be eitherprotein-coding or non-coding. miRNAs also can be transcribed by RNApolymerase III as non-coding RNA transcripts. The primary transcript iscleaved by the Drosha ribonuclease III enzyme to produce anapproximately 70-nt stem-loop precursor miRNA (pre-miRNA), which isfurther cleaved by the cytoplasmic Dicer ribonuclease to generate themature miRNA and antisense miRNA star (miRNA*) products. The maturemiRNA is incorporated into a RNA-induced silencing complex (RISC), whichrecognizes target messenger RNAs through imperfect base pairing with themiRNA and most commonly results in translational inhibition ordestabilization of the target messenger RNA.

Over 650 miRNAs are known in humans and, like transcription factors, asingle miRNA can regulate the expression of numerous genes. This effectgenerally occurs through direct Watson-Crick base-pairing of a small ˜22nucleotide mature miRNA to the 3′ UTR of partially complimentarymessenger RNAs, largely involving the 5′ region of the miRNA known asthe seed sequence. Interaction of the miRNA with cognate messenger RNAstypically results in either destabilization or suppressed translation ofthe messenger RNA targets. Like transcription factors, the spatial andtemporal expression of miRNAs is highly regulated and responsive tochanges in cellular status. See, for example, Ivey & Srivastava, 2010,Cell Stem Cell, 7:36-41; and Martinez & Gregory, 2010, Cell Stem Cell,7:31-5.

miRNA-410 has been identified in a number of mammals (e.g., Homosapiens, Mus musculus, Rattus norvegicus, Macaca mulatta, Canisfamiliaris, Pan troglodytes, Bos taurus, Equus caballus, and Pongopygmaeus), and the mature sequence is 100% identical across species. Themature sequence of mammalian miRNA-410 is 5′-AAU AUA ACA CAG AUG GCCUGU-3′ (SEQ ID NO:1), with the seed sequence located at positions 2-8 ofSEQ ID NO:1.

A modified mature miRNA-410 sequence can be used in the methodsdescribed herein provided that the modified mature miRNA-410 stillretains function (i.e., the modification does not abrogate function).Those of skill in the art would understand that modifications outside ofthe seed region would be the most likely to result in a sequence thatretains function. Modifying a mature miRNA-410 sequence at twonucleotide positions (e.g., outside of the seed sequence) results in asequence having about 90% sequence identity to SEQ ID NO:1, whilemodifying a mature miRNA-410 sequence at a single nucleotide (e.g.,outside of the seed sequence) results in a sequence having about 95%sequence identity to SEQ ID NO:1. Thus, in addition to miRNA-410 (i.e.,SEQ ID NO:1), a nucleic acid having at least 90% sequence identity(e.g., at least 95% sequence identity) to miRNA-410 (i.e., a modifiedmiRNA-410) can be used in the methods described herein.

Percent sequence identity is calculated by aligning two sequences anddetermining the number of identical matches of nucleotides between thetwo sequences. The number of identical matches is divided by the lengthof the aligned region (i.e., the number of aligned nucleotides) andmultiplied by 100 to arrive at a percent sequence identity value. Itwill be appreciated that the length of the aligned region can be aportion of one or both sequences up to the full-length size of theshortest sequence. It also will be appreciated that a single sequencecan align with more than one other sequence and hence, can havedifferent percent sequence identity values over each aligned region. Itis noted that the percent identity value is usually rounded to thenearest integer, and that the length of the aligned region is always aninteger. The alignment of two or more sequences to determine percentsequence identity is performed using the algorithm described by Altschulet al. (1997, Nucleic Acids Res., 25:3389 3402), which is incorporatedinto BLAST (basic local alignment search tool) programs, available atncbi.nlm.nih.gov on the World Wide Web. When utilizing BLAST programs tocalculate the percent identity between two sequences, the defaultparameters of the programs are used.

As used herein, an “isolated” nucleic acid is a nucleic acid that isseparated from other nucleic acids that are usually associated with thereference nucleic acid. Thus, an “isolated” nucleic acid includes,without limitation, a nucleic acid molecule that is free of sequencesthat naturally flank one or both ends of the nucleic acid in the genomeof the organism from which the isolated nucleic acid molecule isderived. An isolated nucleic acid can be introduced into a vector (e.g.,a cloning vector, or an expression vector) for convenience ofmanipulation or to generate a fusion nucleic acid molecule. In addition,an isolated nucleic acid can be an engineered nucleic acid such as arecombinant or synthetic nucleic acid. Isolated nucleic acids can beobtained using techniques routine in the art including, withoutlimitation, recombinant nucleic acid technology, and/or a polymerasechain reaction (PCR). General PCR techniques are described, for examplein PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., ColdSpring Harbor Laboratory Press, 1995. Recombinant nucleic acidtechniques include, for example, restriction enzyme digestion andligation. Isolated nucleic acids also can be chemically synthesized, andalso can be obtained by mutagenesis (e.g., site-directed mutagenesis).

Differentiation of Stem Cells or Downstream Precursor Cells intoPredominantly Astrocytes or Predominantly Neurons and Oligodendrocytes

As this disclosure demonstrates, an increase in the amount or activityof miRNA-410 causes stem cells or downstream precursor cells todifferentiate into a population of predominantly astrocytes. As usedherein, a population that is predominantly astrocytes refers to apopulation of cells that is at least about 55% (e.g., at least about60%, 70%, 75%, 80%, 85%, 90%, 95%, or at least about 99%) astrocytes. Asthose skilled in the art would understand, the amount or activity ofmiRNA-410 can be increased by delivering the miRNA-410 directly to cellsand/or by overexpressing the DNA that encodes miRNA-410 in cells.

As used herein, stem cells or downstream precursor cells refer to cellsthat are able to differentiate into more than one different type ofcell. Generally, in the progression of differentiation, precursor cellsare downstream of (i.e., more differentiated than) stem cells. Forpurposes herein, embryonic stem (ES) cells have multi-lineagedifferentiation capacity and are not restricted to the neural lineage,while neural stem cells and neuronal or glial precursor cells refer tocells that naturally have the capability of differentiating intoneurons, astrocytes, and oligodendrocytes. Neural stem cells usually areidentified by the expression of glial fibrillary acidic protein (GFAP)and nestin and by the ability to differentiate into oligodendrocytes,neurons, and astrocytes. Those of skill in the art would understand thatother stem cells can be used in the methods described herein including,without limitation, induced pluripotent stem cells (iPSC).

miRNA-410 can be delivered directly to stem cells or downstreamprecursor cells using a number of methods routine in the art. Forexample, cells can be electroporated with the miRNA-410 (see, forexample, Andreason & Evans, 1988, Biotechniques, 6(7):650-60), or themiRNA-410 can be introduced into cells using lipid-based deliverysystems, nanoparticle delivery systems, or viral-based delivery systems.See, for example, Patil et al., 2005, AAPS J., 7(1):E61-E77. Inaddition, DNA encoding the miRNA-410 can be overexpressed in cells usingroutine methods. For example, DNA encoding miRNA-410 can be cloned intoan expression vector operably linked to an appropriate promoter, and theexpression vector can be introduced into cells using any of the methodsalready discussed (e.g., electroporation, lipid-based delivery systems,nanoparticle delivery systems, and viral-based delivery systems).

Expression vectors for over-expressing miRNA-410 are commerciallyavailable or can be produced by recombinant DNA technology methodsroutine in the art. An expression vector containing a DNA encodingmiRNA-410 typically will have a promoter (e.g., constitutive orinducible) operably linked to the DNA encoding miRNA-410. Manyconstitutive and inducible promoters are known in the art. As usedherein, “operably linked” means that a promoter and/or other regulatoryelement(s) are positioned in a vector relative to a DNA encodingmiRNA-410 in such a way as to direct or regulate expression of themiRNA-410. It would be understood by those skilled in the art that themiRNA-410 or the DNA encoding the miRNA-410 can be homologous to thestem cells or downstream precursor cells (i.e., from the same mammalianspecies) or heterologous to the stem cells or downstream precursor cells(i.e., from a different mammalian species). An expression vector alsomay include sequences such as those encoding a selectable marker (e.g.,an antibiotic resistance gene).

The amount or activity of miRNA-410 can be increased by increasing(e.g., inducing) one or more of the biological inducers of miRNA-410.For example, bone morphogenic proteins (BMPs) could increase the amountor activity of miRNA-410, thereby increasing the differentiation of thestem cells or downstream precursor cells into astrocytes. As indicatedherein, the methods described herein to increase the amount of activityof miRNA-410 (e.g., direct delivery of miRNA-410, overexpression of theDNA encoding miRNA-410, or induction of miRNA-410) can be used in anycombination. It is expected that using multiple means to increase theamount or activity of miRNA-410 will increase the number of astrocytesrelative to other cells. That is, using multiple means to increase theamount or activity of miRNA-410, it is expected that a substantiallypure population of astrocytes can be produced. As used herein, asubstantially pure population of astrocytes refers to a population ofcells that is at least 95% (e.g., at least 96%, 97%, 98%, 99% or 100%)astrocytes.

As this disclosure also demonstrates, a decrease in the amount oractivity of miRNA-410 causes stem cells or downstream precursor cells todifferentiate into a population of predominantly neurons andoligodendrocytes. As used herein, a population of predominantly neuronsand oligonucleotides refers to a population of at least about 35% (e.g.,at least about 40%, 45%, 50%, 55%, 60%, 65%, 70% or 75%) neurons and atleast about 4% (e.g., at least about 5%, 6%, 7%, 8%, 9%, or 10%)oligodendrocytes. Significantly, by decreasing the amount or activity ofmiRNA-410, the number of oligodendrocytes can be increased three-fold ormore. As indicated herein, the amount of activity of miRNA-410 can bedecreased by increasing (e.g., inducing) one or more of the biologicalrepressors of miRNA-410. For example, noggin can decrease the amount oractivity of miRNA-410, thereby increasing the differentiation of thestem cells or downstream precursor cells into neurons andoligodendrocytes.

As those skilled in the art would understand, the amount or activity ofmiRNA-410 can be decreased by using any number of different methods thathave been developed. For example, antagomirs specific for miRNA-410(see, for example, Krutzfeldt et al., 2005, Nature, 438:685-9),2-O-methyl oligoribonucleotides (2-O-Me-RNA) specific for miRNA-410(see, for example, Berger et al., 2005, In Vitro Cell. Dev. Biol.Animal, 41:12-8), or morpholino oligonucleotides complementary tomiRNA-410 (see, for example, Flynt et al., 2007, Genetics, 39:259-63)can be used. Further, LNA oligonucleotides (see, for example, Ørom etal., 2006, Gene, 372:137-41) or the “miRNA sponge” (see, for example,Ebert et al., 2007, Nat. Methods, 4(9):721-6) can be used to decreasethe amount or activity of miRNA-410 in cells.

The population of predominantly astrocytes, the population ofpredominantly oligodendrocytes, or the population of predominantlyneurons can be used in a variety of methods. For example, thepopulations of predominantly oligodendrocytes, astrocytes or neurons canbe used in drug testing or screening (see below), or the populations ofpredominantly oligodendrocytes, astrocytes or neurons can be used incell therapies for treating diseases associated with a loss, damage ordeterioration of oligodendrocytes, astrocytes or neurons. Diseasesassociated with neuronal loss or damage include, for example,lisencephaly, Rhett's syndrome, hydrocephalus, stroke, and traumaticbrain injury (TBI); diseases associated with oligodendrocyte loss ordamage include, for example, multiple sclerosis, demyelinating disease,leukodystrophies, and agenesis of the corpus callosum; and diseasesassociated with astrocyte loss or damage include, for example, spinalcord injuries, diseases of the blood brain barrier or anomalies of cellmigration such as lisencephaly and hydrocephalus.

Methods of Screening for Compounds that Increase or Decrease the Amountor Activity of miRNA-410

The results described herein regarding the effects of miRNA-410 on stemcells or downstream precursor cells also can be used to screen forcompounds that increase or decrease the amount or activity of miRNA-410(e.g., functional miRNA-410). For example, a nucleic acid molecule canbe produced that includes a promoter operably linked to nucleic acidencoding a miRNA-410 binding site operably linked to nucleic acidencoding a reporter protein. Such a nucleic acid molecule can beintroduced into host cells (e.g., HeLa cells, 293T cells) using routinemethods (e.g., electroporation, lipid-based delivery systems,nanoparticle delivery systems, and viral-based delivery systems), andthe host cells can be contacted with a test compound. It would beapparent to those skilled in the art that an increase in the amount ofreporter protein in the host cells indicates that the test compoundreduces the amount or activity of miRNA-410, and a decrease in theamount of reporter protein in the host cells indicates that the testcompound increases the amount or activity of miRNA-410.

As discussed herein, promoters to drive expression of a DNA sequence arewell known in the art. Promoters suitable for detecting an increase in areporter protein or a decrease in a reporter protein would be known tothose skilled in the art. Reporter proteins, as well as the nucleicacids encoding those reporter proteins, also are well known in the artand include, by way of example, β-galactosidase, luciferase,chloramphenyl acetyltransferase (CAT), green fluorescent protein (GFP)or variants thereof, and mCherry.

In another embodiment, a test compound can be screened for the abilityto increase or decrease the amount or activity of miRNA-410 bycontacting stem cells or downstream precursor cells with the testcompound. Based on the disclosure herein, differentiation of the stemcells or downstream precursor cells into predominantly astrocytesindicates that the test compound increases the amount or activity ofmiRNA-410, and differentiation of the stem cells or downstream precursorcells into predominantly neurons and oligodendrocytes indicates that thetest compound decreases the amount or activity of miRNA-410. For thosetest compounds that do not readily cross the cell membrane, thoseskilled in the art are aware of methods that can be used to introduce acompound into cells (e.g., electroporation, lipid-based deliverysystems, and nanoparticle-based delivery systems).

Test compounds that decrease the amount or activity of miRNA-410 arecandidate compounds for treating diseases in which stimulation ofneurogenesis would be beneficial. Such diseases include, for example,lisencephaly, Rhett's syndrome, and hydrocephalitus. Similarly, testcompounds that increase the amount or activity of miRNA-410 arecandidate compounds for treating diseases in which the production ofastrocytes is stimulated would be beneficial. Such diseases include, forexample, lissencephaly and blood brain barrier dysfunction.

Methods of Treating Tumors

The disclosure herein identifying the role of miRNA-410 in thedifferentiation of stem cells or downstream precursor cells intopopulations that are predominantly astrocytes, oligodendrocytes, orneurons can be used in methods of treating tumors. For example, in oneembodiment, the amount or activity of miRNA-410 can be decreased in amammal having an astroglial tumor. Methods for decreasing the amount oractivity of miRNA-410 are discussed herein and include, for example, theuse of various oligonucleotides (e.g., antagomirs, 2-O-Me-RNA,morpholino oligonucleotides, and LNA oligonucleotides) or a miRNAsponge. Alternatively, a compound identified in a screening method asdescribed herein can be used to decrease the amount or activity ofmiRNA-410 in a mammal having an astroglial tumor.

Representative astrocytic tumors include gliomas, astrocytomas (e.g.,protoplasmic, gemistocytic, or fibrillary), glioblastomas multiforme,pilocytic astrocytomas, subependymal astrocytomas, pleomorphicxanthoastrocytomas, and neurofibromas. Decreasing the amount or activityof miRNA-410 in an astroglial tumor results in differentiation of thetumor into mature, non-proliferating type I or type II astrocytes.

In another embodiment, the amount or activity of miRNA-410 can beincreased in a mammal having a neuronal tumor or oligodendrocyte tumors.Methods for increasing the amount or activity of miRNA-410 are discussedherein and include, for example, direct delivery of miRNA-410 oroverexpression of a DNA encoding miRNA-410. Alternatively, a compoundidentified in a screening method as described herein can be used toincrease the amount or activity of miRNA-410 in a mammal having aneuronal tumor or oligodendrocyte tumors.

Representative neuronal tumors include neuroblastomas, medulloblastomas,retinoblastomas, gangliocytomas, neurocytomas, and ependymoblastomas.Increasing the amount or activity of miRNA-410 in a neuronal tumorresults in differentiation of the tumor into non-proliferating neurons.Representative oligodendrocyte tumors include oligoastrocytomas andoligodendrogliomas. Increasing the amount or activity of miRNA-410 in anoligodendrocyte tumor promotes differentiation of oligodendrocytes.

Any such compound that increases or decreases the amount or activity ofmiRNA-410 can be delivered to a tumor by any number of means including,but not limited to, injection into the tumor, intranasal delivery,intracranial delivery, and delivery via the optic nerve. Delivery of acompound to an astrocytic tumor, an oligodendrocyte tumor, or a neuronaltumor also can be via the cerebrospinal fluid (CSF) by delivery to theperipheral blood stream in combination with one or more agents thatallow such compounds to cross the blood brain barrier, or in vivoelectroporation (see, for example, Anwer, 2008, Methods Mol. Biol.,423:77-89).

In addition to treating tumors, the amount or activity of miRNA-410 canbe decreased at the site of a central nervous system (CNS) injury inorder to inhibit the activation of astrocytes. Since astrocytes exhibita reactive response upon damage to the CNS (e.g., spinal cord injury ortraumatic brain injury (TBI)) and form scar tissue, inhibiting thisactivation of astrocytes by reducing the amount or activity ofmiRNA-410) can provide benefits in treating such injuries (e.g.,ameliorating the damage from such injuries, diminishing the negativeeffects of such injuries, reducing the amount of scar tissue resultingfrom such injuries).

In accordance with the present invention, there may be employedconventional molecular biology, microbiology, biochemical, andrecombinant DNA techniques within the skill of the art. Such techniquesare explained fully in the literature. The invention will be furtherdescribed in the following examples, which do not limit the scope of themethods and compositions of matter described in the claims.

EXAMPLES Example 1 Analysis of miRNA-410 Expression

Microarray analysis identified a microRNA, miR-410, from thesubventricular zone (SVZ) of adult mice. MiR-410 is present in mammals,and the sequence is highly conserved in mouse, human, and multiple othermammalian species. It is present in a cluster of miRNAs (miR379-miR410),one of which (miR-134) is involved in stimulating dendritic outgrowthfrom hippocampal neurons by inhibiting translation of Pumilio2 mRNA.miR-410 was previously identified as expressed in the midgestationembryo, as a candidate nucleic acid for male pattern baldness, and incell cycle control. The structure of the Mus musculus miR-410 is shownin FIG. 1. Using a conditional transgenic mouse in which noggin can beinducibly expressed in nestin+ neural stem cells, it was shown thatmiR-410 was significantly down-regulated relative to a control neuralstem cells when the noggin transgene was overexpressed.

To validate the microarray data, quantitative RT-PCR (qRT-PCR) ofinduced and noggin-overexpressing SVZ was used. It was observed thatmiR-410 was down-regulated more than 15-fold in the induced SVZ (FIG.2). Since miRNAs are short, and family members may differ by a singlenucleotide, care was taken to ensure that only mature miRNA is assayedby PCR. To accomplish this, mature miRNAs were tail-polyadenylated withE. coli poly (a) polymerase prior to reverse transcription. An oligo-dTprimer with a universal primer binding site linked to its 5′ end wasused during reverse transcription. During qRT-PCR, the universal primerand the miRNA-specific primer were employed to ensure that only maturemiRNA was assayed. In situ hybridization analysis were used to confirmoverexpression and inhibition of mature miR-410. Additional microarrayanalysis of RNAs from noggin-expressing and control cerebellar neuralstem cells was performed. It was observed that the miRNA was similarlydownregulated by noggin overexpression.

In situ hybridization localizations of miRNA were performed duringdevelopment and in the adult SVZ. During development, miR-410 wasrestricted to the nervous system, where it was expressed at high levelsat the rhombic lip and midbrain at E11.5 (FIG. 3A). In the adult brain,miR-410 was expressed at high levels in the SVZ neural stem cell zone(FIG. 3B).

To inhibit and overexpress miR-410, two expression constructs weredeveloped (FIG. 4). The backbone of each vector contains a puromycincassette for selection and development of cell lines. The firstconstruct (FIG. 4A) employed the Ubiquitin (Ubc) promoter to driveoverexpression of mCherry and the miRNA. In the second construct (FIG.4B), the Ubiquitin promoter drives expression of a “sponge” in which sixmiR-410 binding sites are expressed downstream of mCherry. BecausemiRNAs are highly conserved and short, and because miRNA familiesdisplay close sequence identities, it is difficult to use smallinterfering RNA (siRNA) technology to target them. Other approachesinclude the use of antagomirs, which are small synthetic RNAs that areperfectly complementary to and which irreversibly bind the target miRNA.For strong, long-term inhibition, a new class of genetically encodedcompetitors of miRNAs was employed: the miRNA “sponge.” These inhibitorsare transcripts expressed from strong promoters, and they containmultiple tandem binding sites specific to the miRNA of interest, thuscompeting with the endogenous targets for the miRNA and de-repressingthe targets.

Example 2 Analysis of miR-410-Expressing Embryonic Stem Cells

To create mouse embryonic stem cell (mESC) lines stably expressingmiRNA-410, D3 mESC were transfected with the expression construct fromFIG. 4A using Lipofectamine 2000. mESC were selected in antibiotic and12 cell lines were expanded and characterized. Two stable cell lineswere selected for analysis, one in which miR-410 is overexpressed 7-fold(line 24) and another in which miR-410 is overexpressed 20-fold (line20) relative to the parental D3 cell lines (see FIG. 5). Additionalcontrol lines expressed mCherry alone. Cell numbers were analyzed after7 days of culture in complete medium. The cells behaved as embryonicstem cells, expressed Nanog and Oct4, and divided rapidly with noevidence of proliferation changes or apoptosis. When grown in neuronaldifferentiation conditions (N2:B27 defined medium with retinoic acid),there was widespread differentiation of Tuj1-positive neurons after 6days in vitro in the control D3 line (FIGS. 6A and 6B), but not in linesexpressing miRNA-410 (FIGS. 6C-D and 6E-F).

To directly address the concern that miR-410 overexpression in embryonicstem cells may promote apoptosis, TUNEL staining was performed in vitro(FIG. 7). There was no significant difference between D3, control lineswith mCherry alone, or in either line 20 or 24. qRT-PCR with lineagerestricted markers was used to identify cells. In addition to theexpected lineages, it is crucial to examine expression of ES markers(Oct3/4), markers of endoderm (FoxA2), and mesoderm (Brachyury) in EScultures. Sox3 was employed for neural precursors and Tuj1 for earlyneurons. Analysis was done on Line 20 cells after 6 days in vitro andcompared with the parent D3 line. When miRNA-410 is expressed 20-foldhigher than in controls, expression of neuronal marker, Tuj1, wasdownregulated 15-fold (FIG. 8).

Example 3 Role of miR-410 in Neural Stem Cells

To examine the role of miR-410 in neural stem cells from the adultbrain, neurospheres were derived from adult SVZ, grown for 14 days insuspension culture with medium containing FGF2 and EGF, and thentransfected with miR-410 expression, sponge, or control constructs. Thevectors were expressed well and many uniformly-shaped red spheres wereobtained (FIG. 9). Spheres were disaggregated and differentiated for 7days in medium lacking growth factors but containing 1% serum.Immunohistochemical localization of cell type-specific antibodies wasthen carried out: GFAP (astrocytes), myelin basic protein(oligodendrocytes), and Tuj1 (neurons).

It was observed that overexpression of miR-410 significantly decreasedthe number of neurons and oligodendrocytes present in these cultures.Astrocyte differentiation was increased, while inhibition using thesponge increased neuronal and oligodendrocyte differentiation to levelsgreater than controls and decreased astrocyte numbers (FIG. 10).Experiments were replicated in three independent experiments, each withthree biological replicates. Cells were counted from five fields in eachreplicate (45 fields).

Quantitative analysis of cell numbers was carried out in differentiatedneurosphere cultures with and without noggin protein. The number of Tuj1positive neurons (FIG. 11, left panel) was significantly increased bymiR-410 inhibition and significantly decreased by the overexpression ofmiR-410. miR-410 overexpression inhibited the differentiation of Tuj1positive neurons produced by noggin while, in the presence of thesponge, noggin significantly increased neuronal differentiation to 60%.

Exposure of neural stem cells to the sponge significantly increased thenumbers of myelin basic protein (MBP)-positive oligodendrocytes comparedto control cultures. These results were sensitive to the presence ofnoggin proteins, either produced by transgene induction or addition ofnoggin protein to the medium.

The number of GFAP positive astrocytes (FIG. 11, right panel) wassignificantly reduced in cells transfected with the miR-410 sponge ascompared with controls. miR-410 overexpression restored thenoggin-promoted reduction in GFAP positive cells to control levels,while, in the presence of the miRNA sponge, noggin significantly reducedastrocyte numbers. In addition, in ES cells, over-expression of miR-410reduces the number of Sox3-positive neural precursors (FIG. 12) andTuj1+ positive neurons (FIG. 13) compared to the parental D3 ES cells.

TABLE 1 Selected Predicted Targets of miR-410 NM_009233 Sox1 Musmusculus SRY-box containing gene 1 (Sox1) NM_001044386 Zfx Mus musculuszinc finger protein X-linked (Zfx), transcript variant NM_001083316Pdgfra Mus musculus platelet derived growth factor receptor alpha(Pdgfra) NM_054043 Msi2 Mus musculus Musashi homolog 2 (Drosophila)(Msi2) NM_172303 Phf17 Mus musculus PHD finger protein 17 (Phf17)NM_009322 Tbr1 Mus musculus T-box brain gene 1 (Tbr1) NM_053242 Foxp2Mus musculus forkhead box P2 (Foxp2) NM_010488 ELAV Mus musculus ELAV(embryonic lethal, abnormal vision, Drosophila)-like 4 (Hu antigen D)(Elavl4) NM_001083967 Tcf4 Mus musculus transcription factor 4 (Tcf4)NM_178880 Son Mus musculus Son DNA binding protein (Son) NM_008702 NLKMus musculus nemo like kinase (Nlk) NM_021543 Pcdh8 Mus musculusprotocadherin 8 (Pcdh8) NM_027642 Phf6 Mus musculus PHD finger protein 6(Phf6) NM_001042660 Smad7 Mus musculus MAD homolog 7 (Drosophila)(Smad7) NM_008008 Fgf7 Mus musculus fibroblast growth factor 7 (Fgf7)NM_008665 Myt1 Mus musculus myelin transcription factor 1 (Myt1)NM_008342 Igfbp2 Mus musculus insulin-like growth factor binding protein2 (Igfbp2) NM_020505 Vav3 Mus musculus vav 3 oncogene (Vav3) NM_144841Otx2 Mus musculus orthodenticle homolog 2 (Drosophila) (Otx2)

Example 4 Effects of Increasing or Decreasing miR-410 on Differentiation

The neural stem cell/subventricular zone (NSC/SVZ) was dissected fromadult mice lacking transgenic noggin gene expression (Uninduced; i.e.,animals carried the noggin transgene but were not exposed to doxicyclineto induce transgene expression), cells were disaggregated and grown intissue culture in the presence of: no additives (+0), the microRNAsponge (+Sp), a miR-410 expression construct (+410), the noggin protein(+Nog), both the sponge and the noggin protein (+Sp +Nog), or both themir-410 expression construct and the noggin protein (+410 +Nog).Additional animals were exposed to doxicycline in vivo to induceexpression of the noggin transgene in the NSC/SVZ (Induced), followed bytissue culture in the presence of: no additives (+0), doxicycline toinduce the transgene in culture (+I), the microRNA sponge (+Sp), themiR-410 expression construct (+410), the noggin protein (+Nog), both themiR-410 expression construct and the noggin protein (+410 +Nog), or boththe sponge and the noggin protein (+Sp +Nog), and differentiation of theneural stem cells into astrocytes (Astros), neurons, or oligodendrocytes(Oligos) quantified. See Table 2 below. The number of astrocytes andneurons were counted and a mean percentage calculated from 45 fieldsfrom three independent experiments with three replicates. The numbers ofoligodendrocytes in the uninduced control cultures was set as 100% andthe effects of manipulations on oligodendrocyte differentiationexpressed relative to control. Table 2.

Uninduced Group. In the presence of the microRNA sponge (+Sp), astrocytedifferentiation was strikingly inhibited with a concomitant increase inneuron and oligodendrocyte differentiation, while over-expression ofmiR-410 (+410) promoted astrocyte and inhibited neuron andoligodendrocyte differentiation. Addition of the noggin protein to thecultures (+Nog) inhibited astrocyte differentiation and promotedneuronal and oligodendrocyte differentiation, which was furtherstimulated by the combination treatment of the noggin protein with thesponge (+Sp +Nog). On the other hand, over-expression of miR-410 in thepresence of the noggin protein (+410 +Nog) was similar to no treatment.

Induced Group. When noggin expression was induced in vivo in the NSC/SVZzone and then neural stem cells obtained and cultured (+0), there was anincrease in neuronal and oligodendrocyte differentiation at the expenseof astrocytes, compared with the uninduced group. When the transgene wasinduced in vivo followed by an additional in vitro induction (+I) thiseffect was augmented, as in cultures exposed to the miRNA sponge (+Sp).When miR410 was over-expressed in induced cells (+410), astrocytedifferentiation was partially rescued, while addition of noggin protein(+Nog) was similar to transgene induction in vivo and in vitro (+I) butslightly less efficient in promoting neuronal differentiation than themiRNA sponge (+Sp). Addition of the noggin protein to miR-410overexpressing cells (+410 +Nog) reverted differentiation to control(+0, Induced) levels, while addition of the noggin protein to cellsexpressing the microRNA sponge (+Sp +Nog) augmented their neuronal andoligodendroglial differentiation.

Overall, the data indicate that miR410 expression or over-expressioninhibits neuronal and oligodendrocyte differentiation, which is rescuedby noggin expression, while expression of the microRNA sponge promotesneuronal and oligodendrocyte differentiation, which can be furtherincreased by the noggin protein.

TABLE 2 Effects of miRNA-410 on Differentiation Mean Mean Mean Mean %Astros ± sd Neurons ± sd Oligos ± sd Oligos Uninduced +0 72.6 ± 1.6 127.3 ± 1.5 2.25 ± 0.4  100 +Sp 58.9 ± 1.6 3 41.1 ± 1.6 5.5 ± 0.2 244.4+410 77.6 ± 6.6 4 22.5 ± 0.9 1.8 ± 0.2 80 +Nog 62.5 ± 1.1 2 37.5 ± 1.14.5 ± 0.2 200 +Sp +Nog 54.2 ± 2.6 5 45.8 ± 2.6 6.7 ± 0.8 297.8 +410 +Nog70.3 ± 1   6 29.6 ± 0.9 2.7 ± 0.8 120 Induced +0 65.3 ± 2.1 7 34.8 ± 2.14.3 ± 0.7 192 +I 55.1 ± 0.5 9 44.9 ± 0.5 6.7 ± 0.5 298 +Sp 53.5 ± 3.1 1046.6 ± 3.1 7.3 ± 1.3 325.8 +410 68.2 ± 0.2 11 31.8 ± 0.2 3.3 ± 0.2 148+Nog 55.2 ± 0.6 8 44.8 ± 0.6 6.7 ± 0.5 298 +410 +Nog 65.5 ± 1.8 12 34.5± 1.8 4.7 ± 0.2 211.8 +Sp +Nog 51.3 ± 0.3 13 48.7 ± 0.3 7.8 ± 0.2 344.4

Example 5 Expression of miR-410 in Glioblastomas

RNAs were isolated from glioblastoma multiforme (GBM) specimens and fromnormal brain from the neurosurgery tumor bank at the University ofMichigan and the amount of miR-410 was determined in qRT-PCR. MaturemiRNAs were polyadenylated prior to reverse transcription. In qTR-PCR,the universal primer and the 410-specific primer were employed to ensurethat only mature miRNA was quantified.

Interestingly, expression of miR-410 was significantly down-regulated inall four GBM samples compared with samples from control brain (FIG. 15).Since GBM tumors contain both neurons and glial cells, GBM tumors likelyrepresent an unusual situation with respect to miRNA-410. Although notbound by any particular theory, the results obtained herein with GBMsamples are likely the result of the preponderance of neurons vs. glialcells in a particular GBM tumor.

Example 6 Luciferase Assays

The goal of these assays is to determine if the candidate target genespredicted in silico, are actually bound by miR-410. To do this, the3′UTR of the target gene was cloned immediately downstream of thefirefly luciferase ORF in the pmirGLO dual-luciferase vector (Promega,Wis.). Firefly luciferase is the primary reporter with an SV40 promoterdriving Renilla luciferase to normalize expression. This construct wasco-transfected with an miR-410 expression plasmid into HEK293 cells thatdo not express miR-410. Controls were vectors containing mutated miR-410binding sites (mt). The expectation is that wild type (wt) reporterswill have less activity than those with mutant sites if they are miR-410targets.

Specifically, the 3′UTR of miR-410 target candidate genes was amplifiedby PCR and cloned into the pmirGLO plasmid between NheI and SalI sitesaccording to the manufacturer's protocol. For genes with a 3′UTR shorterthan 800 bp, the full length 3′UTR was cloned into the vector. For geneswith a 3′UTR longer than 800 bp, a region at least 800 bp longcontaining the miR-410 site in the center was cloned. Vectors containinga 3′UTR with a mutated (mt, dark gray bars in FIG. 16) miR-410 site(TTAATTAA) were made using PCR based site-directed mutagenesis. HEK293cells were co-transfected using Lipofectamine 2000; after 48 h,luciferase activity was measured in a luminometer. The results werestandardized to Renilla expression (to control for transfectionefficiency), with Firefly luciferase activity of controls with a mutatedbinding site set to 100 and wild type luciferase activity expressed as apercentage of control. Data from at least three independent experimentswere then analyzed using Student's t-test.

Of the in silico predicted targets of miR-410, Elavl4, Tcf4, Fgf7 andSmad7 were significantly altered by miR-410, Sox1 was down-regulated atp<0.02, and Zfx and Msi2 were not altered. These data suggest thatElavl4, Tcf4, Fgf7, Smad7 and possibly Sox1 are, in fact, bound andregulated by miR-410. Confirmation by Western blot is performed.Biological function is probed further using over-expression andknock-down of the targets to determine the extent of differentiation ofESC and NSC.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An in vitro method of producing a population ofpredominantly astrocytes comprising: overexpressing miRNA-410 in apopulation of neural stem cells or neuronal or glial precursor cellsand, culturing said cells, wherein said overexpressing miRNA-410 andsaid culturing results in differentiation of said cells into apopulation of predominantly astrocytes, thereby producing a populationof predominantly astrocytes.
 2. The method of claim 1, wherein saidoverexpressing miRNA-410 comprises introducing a nucleic acid moleculecomprising a promoter operably linked to a DNA sequence encodingmiRNA-410.
 3. The method of claim 2, wherein the promoter is aninducible promoter.
 4. An in vitro method of producing a population ofpredominantly astrocytes, comprising: transfecting a population ofneural stem cells or neuronal or glial precursor cells with miRNA-410RNA and, culturing said cells, wherein said transfecting and culturingresults in differentiation of said cells into a population ofpredominantly astrocytes, thereby producing a population ofpredominantly astrocytes.
 5. The method of claim 4, wherein thetransfecting further comprises using a lipid-based delivery system.